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WORLD INTELLECTUAL PROPERTY ORGANIZATION
Ihtemational Bureau

PCX

INTERNATIONAL APPLICATION PUBUSHED UNDER TfclE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification ^
GOIN 21/35

(11) International PubUcation Number: WO 96/41153

(43) International PubUcation Date: 19 December 1996 (19.12.96)

(21) International Application Number: PCT/US96/09304

(22) International Filing Date: 6 June 1996 (06.06.96)

(30) Priority Data:

08/485,366

7 June 1995 (07.06.95)

(71) Applicant (for all designated States except US): INPHOCYTE,

INC. [US/US]; 350 Main Street, White Plains, NY 10601
(US).

(72) Inventor; and

(75) Inventor/Appticant (for US only): ZAKIM, David, S. [US/US];
15 Cole Drive, Armonk, NY 10504 (US).

(74) Agents: KENNARD, Wayne, M. et al.; Hale and Dorr, 60 State
Street, Boston, MA 02109 (US).

(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY,

CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL,
IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV,
MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU,
SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ,
VN, ARIPO patent (KE, LS, MW, SD, SZ, UG), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, DE, DK, ES, n, FR, GB, GR, IE, IT,
LU, MC, NL, PT, SE), OAPI patent (BP, BJ, CF, CG, CI,
CM, GA, GN, ML, MR, NE, SN, TD, TG).

Published

With international search report.

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of
amendments.

(54) Title: BIOLOGICAL CELL SAMPLE HOLDER FOR USE IN INFRARED AND/OR RAMAN SPECTROSCOPY ANALYSIS

(57) Abstract

A biological cell sample holder for use in infrared and/or Raman spectroscopy.
The sample holder includes a rectangular body that has a stepped opening throu^
the center. The body is transparent to infrared and Raman energy. A window is
disposed in the stepped opening. The window has pores of a predetermined size to
sdlow fluid to pass through die window but retain cells of interest on the window.
There also is an assembly that is used to cause the collection and concentration of
cells on the window. The assembly includes a flask with a first outlet that connects
to a vacuum source and a second outlet that connects to a drain system. The flask
has an open top end. A frit sealingly engages the top end of the flask. The fnt is
hollow and has a nipple extending from the top surface of the frit. The frit has a
top surface that is adapted to fit the sample holder.

FOR THE PURPOSES OF INFORMATION ONLY
applica^S ™r*t£ ^S?^ party to the PCX on the front pages of pamphlets publishing international

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BIOLOGICAL CELL SAMPLE HOLDER
FOR USE IN INFRARED AND/OR
RAMAN SPECTROSCOPY ANALYSIS

5 Field of the Invention

The present invention relates to sample holders that are used for holding biological
cells that are to be analyzed by infrared and/or Raman spectroscopy. .
Background to the Invention

Examination of cells and tissues, referred to here as diagnostic pathology, remains

10 a critical step for reaching a medical diagnosis and selecting the most appropriate therapy
for patients. The practice of pathology is limited in reaching definitive diagnoses in many
instances because of the difficulty in identifying morphological changes in individual cells
that correlate with clinical hallmarks of disease. This is an especially significant problem
when cells and not intact blocks of tissue are available for examination.

15 The accuracy and clinical value of microscopic examinations of cells, which may

form a basis for making definitive pathological and clinical diagnoses, is becoming
increasingly important and may provide a method of especially detecting stages of
precancer and cancer without the need for tissue. This method also is attractive because
cells are easier, safer, and cheaper to obtain than tissue, which is available usually via

2Q surgical procedures.

The easy accessibility to cells as compared to tissue makes it possible to use such
cells for screening healthy populations for evidence of early stages of diseases, such as
cancer. Cervical cells, for example, are examined to detect precancer and/or early stages of
cancer of the cervix; cells in urine are examined for evidence of early stages of urogenital
cancer; cells in sputum are examined for early diagnosis of lung cancer. These kinds of

"cytological" tests are becoming increasingly important in the practice of medicine and for
public health. This is true despite the evidence that the clinical value of cytological
examinations is limited and often suspect because of the high incidence of false-negative
results. Cytologic testing also is beset with a high incidence of false-positive results. Both
of these results impact negatively on patient confidence and add unnecessarily to the costs
30 of health care.

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The incidence of cancer is rising as the incidence of other diseases decrease and
people Uve longer. As such, cancer will continue to be a major health problem for years to
come. The best approach to managing the burden of the cancer problem is to find the
disease in its precancerous stages and then to prevem the emergence of frank cancer from
5 precancerous cells. The way to this end is better methods for detecting cells in a

precancerous stage of disease and for showing the extent to which precancerous disease
approaches frank cancer.

An alternative to the traditional method of subjective, microscopic examination of
stained cells for detecting precancerous disease and early stages of cancers is to assess the
chemical and physical properties of the molecules within cells. The logic of this approach
is that normality or abnormality in the chemical and physical properties of the molecules in
cells is the basis for health and disease. Changes in the chemical and physical properties of
molecules in cells precede and underlie the changes in morphology that pathologists search
for microscopically as evidence of disease.

It is known that the vibrational spectra of whole cells, e.g., infrared spectroscopy
15 and Raman spectroscopy, are sensitive methods for measuring whether the molecules in
cells are normal or abnormal. It also is known thai abnormalities in the vibrational spectra
of cells correlate with pathological diagnoses made by microscopic examination of the

tissues and cells. Copending application serial no. , tiUed A System and Method for

Diagnosis of Disease by Infrared Analysis of Human Tissues and CeUs, and filed June 7,
20 1995, demonstrates tiiat infrared spectroscopy of cells detects disease tiiat cannot be

detected by microscopic examination of cells, detects the evolution of normal cells tiirough
tiie continuum of the precancerous changes tiiat eventuate in cancer, detects tiie evolution
of cells through stages of dysplasia tiiat proceeds by different detailed patiiways in ttie
accumulation of genotypic and phenotypic abnomaUties, and detects flie presence of viral
infection of cells.

The use of spectiroscopy for studying cells, i.e., describing in detail how light of
different frequencies interacts witii tiie molecules in cells, is in its infancy as a medical
technology. There is, however, a need for a rapid, inexpensive metiiod for tiie preparation
of cells for examination by vibrational specti-oscopy. Conventional methods of preparing
cells for patiiological examination, e.g., fixing, embedding, and staining of cells, prior to
microscopic examination are not particularly useful for preparing cells for spectroscopic

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examination. Moreover, the methods used by spectroscopists to study inanimate matter
were not useful for preparing cells for vibrational spectroscopy examination for medical
diagnosis. This is because such methods are time consuming, labor intensive, and
expensive. Also, what is being used by spectroscopists for study of cells requires a high
degree of diligence and expertise on the part of the operator, which further inhibits the
general application of the methods of vibrational spectroscopy for the diagnosis of disease.

Principally, there are three known ways to prepare cells for examination by
vibrational spectroscopy. The first is no preparation at all. This method requires that cells
in their natural state be added to a suitable sample holder and analyzed in the presence of
small amounts of water. Second, cells may be placed on a sample holder and any water
removed by drying. Third, cells may be isolated, dried, and incorporated into KBr discs.
Moreover, the method of the direct addition of cells to infrared windows (of BaFj ) by
cytocentrifugation also has been used.

All of these methods have significant problems such as expense, time, and limited
availability of qualified people to do it. However, cells, as they are collected from tissues,
from patients, from the body fluids of patients, from cells in culture, or otherwise, cannot
be used directly in sample preparation method just described.

In order to put the problems in perspective, the specification will consider, for
example, the problem of examining cervical cells by infrared spectroscopy. Cells are
collected from the cervix by scraping with a brush or spatula. For conventional cytology,
20 the cells are smeared directly from the brush or spatula onto glass slides. This method can
not be used as preparation method for vibrational spectroscopy because the beam of light
in the spectrometer cannot cover the area of the typical smear. Moreover, there is difficulty
in controlling the thickness of cells and mucous deposited on the slide. There also may be
some difficulty in fixing cells with materials that can be washed off completely so as not to
interfere with spectral analysis of the cells. Lastly, materials that are used for slide material,
which are transparent to raid-infrared frequencies of light, are relatively expensive.

Rather than smearing, cells can be removed from the collecting brushes and
spatulas by vigorously shaking them in a fluid medium. Next, the cells in the fluid medium
are concentrated and then examined. This concentration is independent of the exact set of
conditions under which spectra will be obtained. If the concentrated cells are examined
directly without drying, the amount of water relative to cells must be quite small or the

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water will detrimentaUy effect the result because of water's avid absorption of infrared
light

As described, cells may be prepared by drying and then examining the dried cells.
Examination of dried cells also cannot be used for vibratory spectroscopy without finally
5 concentrating the cells. Concentration is necessary because only small volumes of cellular
suspensions (in the microliter range) can be added at one time to suitable sample holders
for vibrational spectroscopy. Adding an appropriate number of cells to suitable infrared
sample holders, for example, depends on adding, serially, several microliter acquits of cells
in suspension, allowing each aliquot of the sample to dry on the sample holder before
adding the next aliquot This is a very time consuming process that is not appropriate for
clinical use, i.e., it takes as long as 20 to 30 minutes, for small volumes of sample (about
20 III) to dry.

Even with the dried cells, there will be artifacts unless tiie cells are fixed. Fixation
of cells in this context adds considerable complexity, labor, cost and tiie requirements of
skill and diligence. Fixatives that are contemplated also must be removed from tiie cells by
extensive washing prior to collecting spectra from tiie cells. This step or set of steps cannot
be accomplished within tiie confines of currentiy available sample holders usable for
vibratory spectroscopy.

A sample preparation method tiiat accomplishes concentiation and drying involves
the incorporation of cells into KBr disks. This metiiod depends on die prior concentration
20 of cells followed by drying. Using this method, one gains no advantage over tiie direct
addition of concentrated cells to sample holders.

Concentrating cells may be accompUshed by centrifugation, which isolates tiie cells
from tiie suspending fluid. The concentration of cells is not difficult to achieve by
centrifugation, but it requires speciaUzed equipment time, and labor. Also, tiie need to
25 concentrate cells by centrifugation makes it difficult to automate tiie process of adding cells
to tiie appropriate sample holders. For example, in tiie case of cervical ceUs suspended in
some type of aqueous medium or cells suspended in body fluids, tiie suspension of cells is
centrifuged and tiie supernatant removed by aspiration or decantation. In tiie case of
cervical cells, tiie cells are suspended in a relatively small volume of fluid, which is easy to
remove by centrifugation in small centrifuges, When tiie cells are from body fluids,
however, the volume of fluid can be considerable, e.g., a liter or more, which complicates

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the process of concentration by centrifugation.

Once cells are concentrated as a pellet in the bottom of a centrifuge tube, a small
aliquot of concentrated cells is pipetted directly onto a variety of suitable sample holders.
The cells can be examined in the wet state or the cells can be dried on the sample holder
prior to examination. Examination of cells in the wet state requires a second window for
the sample holder in order to confine the wet sample to a closed system, which increases
costs and labor in cleaning the sample holder.

A complication of examining cells in the dry state, already .stated, is that drying a
sample of 20 to 40 |Jl1 on a sample holder at room temperature requires 20 to 30 minutes.
During this time unfixed cell, autodigest their components, which introduces artifacts into
the spectra collected from such cells.

The problem caused by allowing unfixed cells to stand at room temperature, even
for relatively brief times, is illustrated by the spectra shown in Figure 1. The spectrum in
the solid line is an infrared spectrum of cervical cells collected 10 minutes prior to the
collection of the spectrum in the dashed line on the same sample of unfixed cells on the
same sample holder. Note how the spectrum in the region of 1023 cm"^ was altered by the
metabolic activity of the unfixed cells.

Figure 2 compares two spectra of unfixed cervical cells obtained within a few hours
of each other. Here again, there are significant changes in the spectra of the unfixed cells
over time.

20 Figure 3 compares spectra of cervical cells that were unfixed (dashed line) and fixed

(solid line). The time differential between the unfixed and fixed samples was 30 minutes,
which was the duration of time required to add cells to the window of a convention infrared
sample holder and to dry them at 20^ C. Figure 3 shows how fixation of cells prevents short
term, metabolically-induced changes in the spectral features of unfixed cervical cells.
These examples make plain that the spectral examination of cells is best carried out on fixed
cells because fixing prevents the metabolic function of cells. Otherwise, there is no certain
way to control for the effects of cell viability on spectral features after cells are added to
the sample holder. These examples also demonstrate that in the absence of prior fixation,
heated-drying is not advantageous because heating speeds up the rate of autodigestion of
unfixed cells.

The foregoing provides evidence that conventionally, the examination of human

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ceUs, or ceUs derived from any other source, via infrared spectroscopy is based on
preparing samples individually, one at a time, in a time consuming way. These prior
methods not only require that multiple steps to transfer ceUs from suspensions to a suitable
sample holder but also end up introducing artifacts into the analytical spectra unless tiie
cells are fixed. These methods are expensive, time-consuming, labor intensive, not
amenable to automation, require centiifuges, and depend on die skiU and diUgence of the
operator. Such metiiods also do not lend to tiie rapid preparation of large numbers of
samples at low cost witii minimal skill and diligence by the operator.

Conventional methods for detection of cervical cancer accounts for between
60,000,000 and 80,000,000 examinations of cervical cells each year in the U.S. The
limitations referred to above prevent the full exercise of the capacity of the technology of
vibrational spectroscopy which could replace conventional examinations. In particular, the
limitations of tiie sample holder, loading of the sample holder with cells, and fixation of
these cells remain paramount issues to be solved.

A separate but related difficulty in examining cervical cells, eitiier by standard
cytology or vibrational specti-oscopy, is ttiat the cervix is not a completely homogeneous
organ. For example, it is recognized and recommended that samples of cells be obtained
and examined separately from the endo- and the exocervix. This recommendation is almost
never followed because of tiie economics of carrying out the test. These two samples of
cells per patient require two separate examinations and double tiie real cost of performing
20 the test. Therefore, the method of detecting early stages of cancer of tiie cervix or

precancerous disease of tiie cervix is compromised by taking only one sample tiiat may
contain a mixture of endo- and exocervical cells.

The metiiod of vibrational spectroscopy is not only inherently superior to cytology
as a method for detecting disease in cells, it also is inherentiy cheaper to examine cervical
2^ cells, or other types of cells, tiian standard cytological methods. But tiie difficulty of
preparing samples via conventional metiiods also will compromise tiie total amount of
information tiiat can be collected from cells using infrared spectioscopy. The economics of
preparing samples for spectiroscopic examination, in die absence of better metiiods for
preparing tiiese samples, will dictate tiie collection of only one sample of cervical cells per
patient per examination or ttiat specimens from the endo- and exocervical regions wiU be
combined prior to preparing samples and examining tiieir combined vibrational spectia. As

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such, the method of preparing samples for spectroscopic examination will have a
significant impact on the collection of spectral data and on the amount of clinically useful
information that can be derived from proper sampling of the cells of patients.

There is a need, therefore, for better methods for processing cells from the point of
their collection from patients to their actual analysis by vibrational spectroscopy.
Summary of Invention

The present invention is a biological cell sample holder for use in infrared and/or
Raman spectroscopy. The present invention allows the addition of a suspension of cells and
other components in fluid medium to the window of an infrared sample holder that is
porous and selectively retains cells. According to the sample holder of the present
invention, cells are trapped on the surface of the window while all other components are
filtered through the window. This obviates the need to concentrate cells by some method
independent of placing them on the window. At the same time, trapping the cells on a
porous window makes it possible to wash the cells extensively, treat them chemically in
many different ways, and then to wash away any contaminants that might alter the
15 vibrational spectra. This includes the ability to remove any contaminants that might be
added to the collecting medium to facilitate preparation of the cells.

The present invention requires no change in the manner in which doctors collect
cells from patients. For example, with respect to the cervix, doctors can collect cells by the
method of the current Pap test, by fine needle aspiration of solid tissues, or with respect to
20 other areas, in conventional ways from the sputum, urine, cerebrospinal fluid, ascitic fluid,
pleural fluid, or any other body fluid. Moreover, the present invention also is directly
applicable to the collection of cells in any form that may exist or can be made to exist in a
fluid medium.

The present invention provides a novel method for adding collected cells to suitable
sample holders that hold the cells in an analytical beam of light for the purpose of obtaining
a vibrational spectrum of the cells. The vibrational spectrum can be in any range of the
infrared region and can be obtained by infrared, Raman or resonance Raman spectroscopy.
The present invention also can be applied to collecting spectra by transmission or
reflectance spectroscopy.

The present invention includes sample holder with a window region. Once the cells
to be analyzed are placed on the window region, an analytical light beam is shown through

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cells and the window region. The beam of analytical light must pass without interference
through the window material. The window region is transparent to light of the frequencies
of interest for the analysis to be performed and does not react with components in the
sample holder.

In the present invention, the window region, in addition to the optical requirements
just described, serves the purpose of providing a means for concentrating the material of
interest as it is placed on the window and then a means for treating the sample on the
window in a wide range of different ways, all of which enhance the amount of spectral
information that can be collected from the cells.

Brief Descripti on of the Drawings

Figure 1 shows a first comparison of spectral waveforms for unfixed ceUs.
Figure 2 shows a second comparison of specUral waveforms for unfixed cells.
Figure 3 shows a comparison of spectral waveforms for fixed and unfixed ceUs.
Figure 4A shows a top view of the sample holder of the present invention.
Figure 4B shows a cross-sectional view of the sample holder of the present
invention at 4B-4B of Figure 4A.

Figure 5 A shows the vacuum filtration system of the present invention.

Figure 5B shows the vacuum filtiration system of Figure 5 A with the sample holder
of the present invention mounted on it.
20 Figure 6 shows a fluid suspension containing collected cells in a syringe with the

fluid suspension being added to the sample holder of the present invention.

Figure 7 shows the sample holder of the present invention with a detachable funnel
connected to it.

Figure 8 shows a second embodiment of the system of the present invention for
2^ collecting and concentrating cells.

Figure 9 shows a top view of the frit in the second embodiment of tiie system shown
in Figure 8.

Figure 10 shows a top view of the window and non-porous transport support.
Figure 1 1 A shows a top view of a second embodiment of a sample holder of the
present invention.

Figure 1 IB shows a bottom view of the second embodiment of the sample holder

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of the present invention.

Figure 12 shows an assembly for removing cells from collecting devices.

Figure 13 shows a third embodiment of the system of the present invention for
collecting and concentrating cells.
5 Figure 14 shows a system and method for automatic or semi-automatic collection

and concentrating cells.
Description of the Invention .

The present invention is a biological cell sample holder for use in infrared and/or
Raman spectroscopic analysis. The present invention principally will be described in the
context of processing cervical cells for analysis by vibrational spectroscopy. However, it
is understood that the present invention applies to any type of cell or source of cells. For
example, cells from any body fluid, including blood can be processed in the same manner
as cervical cells. Further, cells in experimental systems, e.g., cells in culture, whether
human cells, animal cells, plant cells, normal cells, and diseased cells, can be processed by
the present invention,

15 A top view the sample holder of the present invention is shown in Figure 4 A. Figure

4B is a cross-sectional view of the sample holder of the present invention at 4B-4B of
Figure 4 A. Referring to these Figures, body 102 of sample holder 100 serves as a structure
upon which a sample may be placed. Body 102 has stepped opening 107 located in the
center. Stepped opening 107 has upper section 108 and lower Section 110. Annular ledge

20 1 12 is formed between the two sections. Window 104 is disposed in the opening and is
supported by annular ledge 112.

Samples to be analyzed are placed on Window 104. Analytical light illuminates the
cells on the window to obtain spectral information. Window 104 of sample holder 100 is
transparent to predetermined frequencies of light. Moreover, the window is porous so that
water and materials dissolved in water will pass through it. The pores are not large enough,
however, to permit passage of cells. In fact, the pores of the window will allow fluid to pass
through the window at pressures that will not rupture window 104 or tear it from body 102
of the sample holder 100.

Body 102 also includes groove 106 disposed concentric with stepped opening 107.
Groove 109 is for the attachment of a funnel (not shown) that is used for collecting cells

^® from large volumes of fluid material, as will be described. Groove 1 16, however, is not

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required to practice the present invention.

Referring to Figure 5 A, the vacuum filtration system of the present invention is
shown generally at 150. Vacuum filtration system 150 is used for loading cells in
suspension onto window 104. Vacuum filtration system 150 includes vacuum flask 152 and
5 fidt 164 that is disposed in opening 154 at the top of vacuum flask 152.

Vacuum flask 152 has vacuum outiet 156 which is connected to a vacuum pump (no
shown) that will draw a predetermined level of vacuum in vacuum flask 152. Vacuum flask
152 also has drain outiet 158 to which drain line 160 connects. Valye 162 is disposed in
drain line 160 to control fluid drainage from vacuum flask 152.

Frit 164 has an outside contour and shape that permits it to sealingly fit in top
opening 154 of vacuum flask 152. Frit 164 has top surface 166 and opening 170 in the
bottom. Hollow nipple 168 extends upward from top surface 166. Hollow nipple is in fluid
communications witii opening 170. Frit 164, preferably is made from sintered glass.

Referring now to Figure 5B, vacuum filtration system 150 is shown with sample
holder 100 disposed on it. As is shown, hollow nipple 168 is dimensionally shaped to fit
into lower section 1 10 of stepped opening 107 in body 102 of sample holder 100, and up
against the bottom of window 104.

Even though the Figures 5A and 5B show frit 1 64 with hollow nipple 168 extending
from up surface 166, tiie present invention contemplates other configurations of frit 164,
which includes without limited a flat frit with an opening to accommodate ttie size of
20 window 104 and window 104 is disposed flush with the bottom of body 102.

Again referring to Figures 5A and 5B, constructing frit 164 to tiie exact contour of
body 102 and window section 110 of tiie sample holder 100 insures efficient application of
suction pressure through window 104. As such, negative pressure tiiat is applied tiirough
vacuum flask 152 does not rupture tiie window or tear it from body 102. Therefore, all tiiat
25 happens when the vacuum is applied through causing suction tiirough vacuum outiet 156
is window 104 of sample holder 100 is drawn tightiy to the surf-ace of tiie sintered glass frit
164.

Cells in suspension may be added to tiie center of tiie window by any convenient
metiiod such as by a pipette (not shown). The existence of upper section 108 of stepped
opening 107 prevents tiie wetting of body 102 of sample holder 100 as suspensions of cells
are added to window 104.

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As the cells in a fluid medium are added to window 104, the fluid medium and
dissolved components are drawn through the pores of window 104 by vacuum pressure.
Since the pores of window 104 have the appropriate size, the cells become trapped at the
surface of window 104. The fluid suspension is added to window 104 at a rate that will
permit the fluid medium to filter through window 104 and collect in vacuum flask without
wetting the sample holder. This novel configuration allows for the simultaneous
concentration and addition of cells to window 104. Accordingly, there is no need to
concentrate the cells in the suspension prior to adding them to window 104 of a sample
holder 100.

Another aspect of the vacuum filtration system 1 50 is that the simultaneous addition
of cells to window 104 and drying of such cells is accomplished by application of negative
pressure to flask 152. As such, window 104 of sample holder 100 facilitates the processing
of cells for spectral examination by rapidly and inexpensively collecting and concentrating
the cells. Once cells have been added to window 104 of the sample holder 100, the cells are
analyzed using infrared and/or Raman spectroscopy.
15 Referring to Figures 4A, 4B, 5A and 5B, aspects of sample holder 100 will be

described in greater detail. Body 102 of sample holder 100 preferably is constructed of a
molded plastic. However, it is understood that other methods may be used. For example,
body 102 of sample holder 100 could be constructed of paper or cardboard, or other suitable
material. Window 104 may be constructed of any suitable material that has the necessary
20 optical properties for vibrational spectroscopy. This material also must be porous. The
upper limit to the pore size must be less than the diameter of cells. Examples of suitable
materials for window 104 are microporous non-woven or fibrous webs of glass,
polyethylene, polypropylene, and alumina.

Window 104 may be thin for some application and thicker for others. The thickness
depends on the optical limitations imposed by the type of analysis that is to be conducted
and the nature of the material being analyzed. For example, windows constructed of
polyethylene have strong vibrational bands in the mid-infrared region. If such windows are
relatively thick in the 2-3 mm ranges, they will not transmit enough light in the mid-
infrared to be useful. However, if these materials are thin sheets in 10 to 20 |Jim range, they
are excellent windows for infrared spectroscopy of cells and tissues. By contrast, thick
^® layers of glass, as windows, do not create any problem for near infrared spectroscopy, or

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Raman or resonance Raman spectroscopy.

Before the cells are analyzed, they may be washed to remove any materials which
will impact negatively on the spectral response from the cells or materials under analysis.
Washing of the ceUs may be accomplished by using a pipette of wash solution of any
5 volume. The washing step is repeated until the undesired materials are washed from the
cells.

The fixation of cells provides a method to obtain a better response from the
vibrational spectroscopic analysis of cells. Fixation is not used primarily in structures in
which spectral examinations are conducted immediately after the coUection of cells,
otiierwise fixation is a preferred method. However, in an automated system of analysis, in
which cells from several samples are prepared and allowed to stand prior to examination
by a spectrometer, e.g., in large central laboratories, fixation is used to assist in securing the
successful use of spectroscopic methods to large numbers of samples.

Anotiier method of processing the cells to be analyzed is to freeze them until they
are prepared for examination by vibrational spectroscopy. Once unfrozen tiie cells are then
prepared and maintained atlow temperatures until the time of analysis. This, however, adds
enormous complexity and expense to tiie infrared or Raman spectroscopic examination of
cells and tissues.

In cases when a fixative material is added to the cells, it is important to remove the
fixative prior to spectral examination so fliat artifacts based on tiie fixative are not present.
20 The fixative may be removed by washing cells as extensively as desired once ttiey are
trapped on window 104 of sample holder 100. Specifically, the fixative is removed by
washing window 104 with appropriate solutions.

In collecting of cervical cells, the cells are scraped from tiie cervix witii brashes and
spatulas. In preparing standard cytological smears, die cells attached to tiie brushes and/or
23 spatulas are smeared onto glass slides. For tiie purpose of specti-al analysis, however, tiie
collecting devices are placed in capped botties containing a buffered salt solution plus a
fixative. Vigorous shaking of ttie sealed botties displaces tiie cells from the brushes and
spatulas and suspends the cells in tiie fiuid in tiie collecting botties. The suspension of cells
can be aspirated from tiie botties and added to window 104 of sample holder 100. The ceUs
are fixed when tiiey are removed from tiie botties in which tiiey are collected.

Cells collected by fme needle aspiration from tiie botties are aspirated into a fluid-

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filled syringe. The cells collected in this manner can be added directly to window 104 of
sample holder 100. This may be done within seconds of cell collection. This action is
shown in Figure 6 at 200.

In Figure 6, fluid 208 from syringe 202 is expelled from reservoir 204 through
5 needle 206 upon positive pressure on plunger 210 of syringe 202. Cells can be fixed once
they are placed on window 104 of sample holder 100 to avoid an intermediate step of
adding the cells to fixative and then adding the suspension of cells in fixative to window
104. Preferably, fixation is accomplished by the addition of fixatiye to window 104 of
sample holder 100. The vacuum is turned off to control the duration of contact between the
fixative and the cells. After this period, the vacuum is turned on to remove the fixative. The

excess fixative is then washed from the cells under vacuum by an appropriate wash
solution. Also, once fixation is complete a series of washing steps take place to remove
fixative from the cells.

The present invention facilitates the addition of cells to window 104 of sample
holder 100 in a manner to speed up and simplify sample preparation for spectral analysis.

1 5 The present invention also enhances the user' s ability to remove biological substances from
sample holder 100 that are of no interest spectrally or that might interfere with the spectral
analysis of the cells. This could be, for example, the desire to diminish the amount of
mucous in the sample on window 104, The user could do this by repeatedly washing the
cells trapped on window 104 with large volumes of water or with a solution of normal

20 saline. An alternative to simply washing away "contaminating materiaF' of no spectral

interest or material that might confound the spectra'of the cells is to wash the window with
chemical mixtures that react with contaminants. For example, again in the case of the desire
to remove mucous, the cells trapped on the window can be washed with mucolytic agents
to remove this mucous. In this regard, the contact time between wash liquid and cells
trapped on window 104 can be controlled by varying the strength of the applied vacuum;

this is especially valuable when the body is nonwettable as in the case of polyethylene,
polypropylene, or other suitable hydrophobic materials.

According to the present invention, the preparation of the cells trapped on window
104 may be modified for desired purposes. Solutions containing vibrationally useful probes
of surface molecules can be reacted with cells trapped on the window and then washed
away prior to spectral analysis of the cells. This will provide means for enhancing or

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suppressing desired spectral aspects of the cells.

Also according to the present invention, it is possible to remove small cells from
large ceUs on window 104. Samples of cervical cells often contain blood cells, which are
quite small as compared with the size of cervical epitheUal cells. Proper selection of the
5 pore size in window 104 will allow for the separation of epitheUal cells from blood cells.
This wUl enhance the spectral analysis of the small numbers of epithelial cells in tiie
presence of large numbers of contaminating blood cells.

The above examples apply to processing cells in relatively small volumes of body
fluids, e.g. 1 to 2 ml or less. The present invention applies equally to processing cells in
exttemely dilute suspension in large volumes of body tluids. This is true even when tiiere
are titer amounts of fluids such as urine, ascites, pleural tluid, and cerebrospinal fluid, and
the like in which a small number of cells reside. These larger fluid amounts can be easily
added directiy to window 104 of sample holder 100 without prior treatment of the cells or
efforts to concentrate tiiem in any way. The entire volume of fluid up to several liters, and
all the cells suspended in a large volume, can be added to window 104 of sample holder
15 100. as will be described.

Referring to Figure 7, generally at 260, when large volumes of dilute suspensions
of cells are to be processed, tiie sample holder and vacuum filtration system include
detachable funnel 262 tiiat is disposed in groove 106 in the top surface of body 102 of
sample holder 100. Funnel 262 has bottom edge 208 tiiat is dimensioned to fit into groove
20 106 in body 102. Spaced up from bottom edge 208 is circular flange 266 which is used for
handUng funnel 262. Funnel 262 prevents spillage and loss of cells.

Figure 8 at 300 shows a second embodiment of the system for adding and
concentrating cells on window 304 for vibrational spectroscopic analysis. In Figure 8,
window 304 is not attached permanentiy to body 308 of filter holder 302 but is free and is
25 inserted into filter holder 302. Window 304 is disposed wifliin filter holder 302 and filter
holder 302 can be opened after use to remove window 304. FUter holder 302 may be made
of any suitable material.

Preferably filter holder 302 includes frit 308 and cap 3 14. The frit and cap are made
preferably from molded plastic. Cap 314 has a hollow member tiiat extends upward from
tiie top. Cap 314 and frit 308 sealably and detectabiUty mate. Filter holder 302 holds
window 304 in place and prevents it from tearing when positive pressure is appUed.

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Referring to Figure 8, window 304 is bordered by non-porous frame 312 that is
disposed on frit 308. Frame 312 restricts the cells collected on window 304 to small area
and facilitates manipulation of window 304 after it is loaded with cells.

Figure 9 shows a top view of frit 308. Frit 308 is disposed below window 304 and
has a plurality of openings 310 in fluid connections with the flask 152.

The member that extends upward from the top of cap 314 connects to syringe 320
via Luer lock 322 or any other suitable attachment mechanism. Plunger 324 is disposed in
the top end of syringe 320 for forcing fluid with cells down to window 304. More
specifically, the cells suspended in fluid medium within the barrel of the syringe 320 are
collected on the window by applying positive pressure to plunger 324 of syringe 320 and
filtering the suspension through window 304 that is held in filter holder 302. Once this is
accomplished, window 304 with attached cells is removed from filter holder 302 by
grasping window 304 via non-porous border 3 12. Filter holder 302 is discarded if it is made
of plastic or other inexpensive materials or it can be washed for reuse if it is made of
stainless steel or other expensive material.
15 Window 304 with trapped cells is mounted on body 352 of disposable sample

holder 350 that is shown in Figures 1 1 A and 1 IB. Figure 1 1 A shows the top view and
Figure 1 IB shows the bottom view of the sample holder. Other methods for mounting
window 304 on body 352 of sample holder 354 can be used. For example, window 304 may
be mounted magnetically to a steel sample holder.
20 Window 304 with trapped cells is removed from filter holder 302 and placed in

infrared transparent support 380. This transparent support shown in Figure 10, preferably
is made from crystalline CaFj or other crystalline, infrared-transparent materials. Window
304 and support 380 may be mounted in standard infrared sample holder 354.

Referring to Figure 12, an assembly is shown for removing cervical cells from
brushes or spatulas. With plunger not removed from barrel 402 of modified syringe 400,
the brush or spatula with cells attached is placed in the fluid medium in the barrel. Cap 406
is placed on the top of barrel 402 and the assembly is shaken to dislodge the cells from the
collecting devices into the fluid medium. Cap 406 is removed and the collecting devices
are removed from barrel 402. Next, the syringe is fitted with plunger 404. Plunger 406 is
used to apply positive pressure on the fluid medium containing the cells.

A third embodiment of the system for collecting and concentrating cells is shown

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in Figure 13. This embodiment has an attached metal channel on cap 314 for puncturing the
bottom of syringe 400 (Figure 12). Positive pressure on plunger 404 of the syringe 400 will
cause cells in the fluid medium to be appUed to window 304. Alternatively, cervical cells
in suspension, or any other type of cell in any type of collecting tube, are aspirated into a
5 standard syringe. The standard syringe then is attached to filter holder 302 as in Figure 8,
and the cells in suspension are added to the window held within the filter holder.

Any of tiie manipulations of cells described with respect to embodiment of tiie
invention depicted by Figures 4A. 4B, 5A and 5B can be applied to the embodiments in
Figures 8 -13. For example, cells can be fixed after they are trapped on the window within
tiie filter holder by changing syringes and treating tiie window with appropriate fixative.
The filter holder and syringe systems shown in Figures 8-13 can be used witii fixed cells.
When fixed cells are used, residual fixative is washed from the cells by changing tiie
syringe to appropriate wash solution. Additionally, fixative or otiier reagents of potential
use in tiie analysis of the cells can be present in tiie syringes to which cells are added.

Referring to Figure 14 at 500, tiie system and method (automatic and semi-
automatic) of the present invention will be described. After vigorous shaking to ttansfer
cells from collecting devices to fluid suspension 552 in tiie collecting device 502, tiie
system shown generally at 500 is used for manual or automatic collection and concentrating
of cells for analysis. First end 509 of ftibe 506 is disposed in fluid suspension 552 in
collecting device 502. Pump 516 causes tiie fluid medium to flow in direction "A" is tube
20 506. The cells are delivered to window 532 of sample holder 530 from end 507 of tube 506.
The vacuum suction in flask 520 draws tiie fluid associated with tiie cells tiirough tiie
window and frit into the flask. This action may take place automatically. Preferably, tiie
rate of aspiration for automatic operation substantially matohes tiie rate of ftotion, or ttie
former is slower tiian the latter, to prevent spillage or waste of cells. All or flie part cells
25 may be added to window 532 in tiiis way. The amount of ceUs to be added can be conti-oUed
by a device tiiat controls the volume of tiie aspirate. Even tiiis feature of tiie device can be
controlled automatically, which is especially useful for controlling tiie amount of cells
added to the window.

According to Figure 14, tiiere is continuous monitoring tiirough a suitable optical
window 512 of ttie aspirated stteam in flie flow patii between aspirating pipette 506 and
window 532 of sample holder 530. Window 512 may be observed visually or via tiie

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sensors at 540 and 542. The simplest method, in this regard, and the preferred embodiment
of the present invention is to monitor turbidity by light scattering. Other ways to monitor
the amount of cellular material in the stream are by absorption of protein or DNA, for
example, but light scattering will be intense at short wavelengths. Independent of the
optical method used to monitor the amount of cellular material in the stream of flow to
window 532 of sample holder 530, a built-in program correlates flow rate for aspiration and
the measurement of turbidity (or another optical property) to calculate the volume of
suspension that must be added to window 532 to yield a sample with an optimal number of
cells on window 532. When this volume of cells is added, aspiration is cut-off.

To facilitate a uniform distribution of cells in suspension, after shaking to displace
them from the collecting devices, the collecting fluid is maintained at high density by
addition of sucrose or other suitable material. Whatever material is used to increase the
density of the collecting fluid and thereby to decrease the rate of settling of cells, it must be
washed from the window after the requisite number of cells (based on measurements of
turbidity) has been added to window 532. In the case that an inadequate number of cells has
been added, after aspirating all the suspension, the system can provide a print out, a flashing
light, or other suitable alarm (not shown) to show this condition.

In a large clinical pathology laboratory, complete automation of sample preparation
can be obtained. In such operations, the bottles containing the cells are shaken
automatically. Two sample holders, one for the endo- the other for the exocervical samples,
are imprinted automatically with the same identifier code. Then the suspensions of endo-
and exocervical cells are aspirated and added to the appropriate windows. Control of the
number of cells added to each substrate is effected as described above and illustrated in
Figure 14. Once the cells are added, routines are carried out for washing cells and/or for
treating the cells with fixatives, and for specific chemical treatments for specific
modification of the cells, as described.

In Figure 4, window 104 of sample holder 100 preferably has a diameter of
approximately 3 mm. The beam of light in a standard infrared spectrometer is reduced to
as small as 1.3 mm. With microscopic attachments, smaller light beams can be
accomplished, down to the limit of diffraction effects. In the mid-infrared, the diffraction
limit is greater than the dimensions of a single cell. In the near infrared, however, light
beams the size of a cell can be produced, which applies as well to spectroscopy by Raman

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scattering.

Generally, spectra collected on cells represent an average spectrum of all cells in a
sample. The capacity of vibrational spectroscopy to detect disease in cells and stage the
severity of disease can be maximized by examining cells one at a time. To do so, requires
that cells be added to a window such that the cells are spread out across the surface of the
window. According to the present invention, the application of cells to the window
provides a conttolled method of adding cells to the window so that vibrational spectra can
be collected simultaneously on a minimal number of cells at any one time or even one cell
at a time. To do this, tiie aspirated ceUs in suspension are added to the window as shown in
Figure 14.

The drop-wise addition is controlled, as regards the size of the drops (which depend
in part on the concentration of cells in the suspension) and their location on tiie window, by
computer software that drives tiie tip of the pipette in small increments (as small as 1 ^m,
for example) across the horizontal and vertical dimensions of tiie window. Since flie
amoum of fluid added at any one time is small and not allowed to spread across tiie window,
tiie cells become "stuck" where they are deposited. The coordinates of tiie window at which
cells are deposited are then used as a basis for washing the cells, again in drop-wise fashion
or for treating cells as discussed above. Finally, the coordinates of location of cells on tiie
window are fed on-line to tiie spectrometer, which has microscopic optics. The
spectiometer collects and co-adds interferograms from each coordinate moving across and
20 sampling multiple regions of the surface of tiie window. Software may direct tiie collection
in this way on tiie basis of tiie stored coordinates for tiie positions of cells on tiie window.
Each co-added spectrum is analyzed separately and simultaneously with tiie scanning of ttie
window until the entire window has been scanned - cell by cell in some cases - or until a
definitive diagnosis of disease can be made.
25 The terms and expressions which are used herein are used as terms of expression

and not of limitation. There is no intention in the use of such terms and expressions of
excluding tiie equivalents of tiie features shown and described, or portions tiiereof , it being
recognized tiiat various modifications are possible in ttie scope of the present invention.

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Claims:

1. A sample holder for collecting and concentrating cells for use in vibrational
spectroscopy, comprising:

a central body having a predetermined shape and being transparent to infrared and
Raman energy;

a stepped opening through the body:

a window disposed in the stepped opening, with the window being transparent to
infrared and Raman energy and including pores of predetermined size extending through
the window.

2. A system for collecting cells on a window of a sample holder that is used in
vibrational spectroscopy:

a container having a first outlet for connection to a vacuum source, a second outlet
for connection to a drain means, and a top end; and

an interface member that sealingly connects to the top end of the container and has
a surface adapted to mate with the sample holder, with the interface member being in fluid
communications with both the window of the sample holder and the container.

3. A system for collecting cells for use in vibratory spectroscopy, comprising:

a sample holder for collecting and concentrating cells, with the sample holder fur-
ther including,

a central body having a predetermined shape and being transparent to infra-
red and Raman energy;

a stepped opening through the body:

a window disposed in the stepped opening, with the window being trans-
parent to infrared and Raman energy and including pores of predetermined size extending
through the window; and

an assembly for causing cells to be collected and concentrated on the window of
the sample holder, with the assembly including,

a container having a first outlet for connection to a vacuum source, a sec-
ond outlet for connection to a drain means, and a top end; and

an interface member that sealingly connects to the top end of the container

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PCT/US96/09304

and has a surface adapted to mate with the sample holder, with the interface member being
in fluid communications with both the window of the sample holder and the container.

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1000 1200

WAVEHUHBER (I /cm)

FIG, 2

SUBSTITUTE SHEET (RULE 26)

wo 96/41153

PCT/US96/P9304

2/6

1000 1200
WAVEHUHBER (I/cm)

FIG. 3

FIG, 4 A.

106

108 ^ft-/^/

WW.

112

I/O

-102

SUBSTITUTE SHEET (RULE 26) ,

wo 96/41153

PCT/US96/09304

3/6

FIG, 5 A

:SUBSTITUTE SHEET (RULE 26)

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PCT/US96/09304

SUBSTITUTE SHEET (RULE 26)

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PCT/US96/09304

5/6

■262

-260

266

208
102

FIG. 7

FIG, 9

^350

^352

FIG, 11 A

^350
-352

FIG, IIB

suBsrrruTE sheet (rule 26)

wo 96/41153

PCT/US96/09304

6/6

404

400

402

406

\s — >]

FIG. 12

-302

•304

FIG, 13

FIG . 14

SUBSTITUTE SHEET (RULE 26)

INTERNATIONAL SEARCH REPORT

Inte onal Application No

PCT/US 96/09304

A. CLASSIFICATJON OF SUBJECT MATTER

IPC 6 G01N21/35

According to International Patent aassification (IPQ or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 6 G01N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search teims used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category '

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US, A, 5 408 306 (ANDERSON) 18 April 1995
see abstract

see column 4, line 49 - line 60
see column 5, line 30 - line 40
see column 5, line 45 - line 51
see figures 3,4

US, A, 3 521 963 (BADER) 28 July 1970
see column 4, line 41 - line 48
see column 4, line 55 - line 61
see figures

WO,A,90 15981 (UNIVERSITE CATHOLIQUE DE

LOUVAIN) 27 December 1990

see page 1, line 7 - line 18

see page 7, line 22 - line 26

see page 8, line 9 - line 13

see figures 9,10; examples 6,7

-/" '

1,3

1-3

Further documents are listed in the continuation of box C.

Patent family members are listed in annex.

* Special categories of cited documents :

'A' document defining the general state of &e art which is not
considered to be of particular relevance

'E' eaiiier document but published on or after the international
filing date

'L* document which may throw doubts on priority claim(5) or
which is dted to establish the publication date of another
citation or other special reason (as specified)

'O' document referring to an oral disclosure, tise, exhibition or
other means

'P* document published prior to the international filing date but
later than the priority date claimed

"T" later document published after the international filing date
or priority date and not in conflict with the application but
dted to understand the principle or theory underiying the
invention

'X' document of particular relevance; the daimed invention
cannot be considered novd or cannot be considered to
involve an inventive step when tiie document is taken alone

"Y* document of particular relevance; the daimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.

*&* document member of the same patent family

Date of the actual completion of the international search

30 September 1996

Date of mailing of the international search report

Qf^ Ttt 96

Name and mailing address of the ISA

European Patent Ofllce, P.B. SSI 8 Patentlaan 2
NL - 2280 HV Rijswijk
Td. (+ 31-70) 340-2040, Tx. 31 651 cpo id.
Fax: (+31-70) 340-3016

Audiorized officer

Thomas, R.M.

Fom PCT/ISA/210 (second sheet) (July 1993)

page 1 of 2

INTERNATIONAL SEARCH REPORT

Intel mal Application No

PCT/US 96/09304

C.(Continiiation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category '

Citation of document, with indication, where appropriate, of the relevant

Relevant to claim No.

W0.A.93 00580 (MINNESOTA MINING) 7 January

see abstract
see page 20, line 34
claim 30

1-3

page 21, line 21;

Fotm PCTASA/aO (ooati

of ncond Micm) (July 19*2)

page 2 of 2

INTERNATIONAL SEARCH REPORT

Infonnation on patent family members

Intcv Jnal Application No

PCT/US 96/09304

Patent document

Publication

Patent family

Publication

cited in search report

date

member(5)

date

US-A-5408306

18-04-95

NONE

US-A-3521963

28-07-70

NONE

WO-A-9015981

27-12-90

DE-D-

69004430

09-12-93

DE-T-

69004430

tm mm 0% M Ji

17-03-94

EP-A-

Ail "JQCnC

04/oDyo

08-04-92

JP-T-

4506402

05-11-92

WO-A-9300580

07-01-93

AU-A-

2304592

25-01-93

CA-A-

2103446

26-12-92

EP-A-

0591417

13-04-94

JP-T-

7500180

05-01-95

US-A-

5470757

28-11-95

Fonn PCT/lSA/aiO (pstant funtty annex) <July 19»)

(19) 02f:S#fF;f (J P)

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¥^£9^(1997)12^ 8 B
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Full text of "USPTO Patents Application 10561839"

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